小柯机器人

结构解析Prp5如何校对前体信使RNA（pre-mRNA）的分支位点，这一成果由德国马克思普朗克生物物理化学研究所Reinhard Lührmann和Holger Stark研究组经过不懈努力而取得。相关论文发表在2021年8月4日出版的《自然》杂志上。

通过删除分支位点腺苷(BS-A)或突变肌动蛋白前体mRNA的分支位点序列，研究人员在A复合体形成之前直接阻断了酿酒酵母提取物中剪接体的组装。然后，研究人员利用冷冻电镜解析了这一新发现组装中间体的三维结构。结构分析表明U2-BS螺旋已经存在于该pre-A复合体中，但尚未被Hsh155蛋白(Hsh155^HEAT)的HEAT结构域夹住，Hsh155^HEAT呈现开放构象。该结构进一步揭示了在A复合物形成期间 U1和U2小核核糖核蛋白(snRNP)的大规模重构/重新定位，这是U4/U6.U5三-snRNP结合所需的，但这种重新定位是由含Prp5的pre-A复合物介导完成。

该研究的数据表明，Hsh155^HEAT与U2-BS螺旋凸起的BS-A结合导致了 Hsh155^HEAT构象闭合，这反过来又破坏了Prp5的结合。因此，如果分支位点突变阻止了Hsh155^HEAT的重构，Prp5会间接校对分支位点，从而阻碍剪接体组装。这些数据提供了关于剪接体解旋酶如何增强前体mRNA剪接保真度的结构证据。

据了解，在pre-mRNA内含子剪接过程中，U2 snRNP必须稳定整合到剪接体A复合体中，这一个由DEAD-box解旋酶Prp5调控的多步骤过程，但对于该过程的机制知之甚少。在这个过程中，U2小核RNA (snRNA) 与前体mRNA分支位点（U2-BS螺旋）形成RNA双链体，但该阶段由Prp5介导的校对机制也尚不可知。

附：英文原文

Title: Structural insights into how Prp5 proofreads the pre-mRNA branch site

Author: Zhang, Zhenwei, Rigo, Norbert, Dybkov, Olexandr, Fourmann, Jean-Baptiste, Will, Cindy L., Kumar, Vinay, Urlaub, Henning, Stark, Holger, Lhrmann, Reinhard

Issue&Volume: 2021-08-04

Abstract: During the splicing of introns from precursor messenger RNAs (pre-mRNAs), the U2 small nuclear ribonucleoprotein (snRNP) must undergo stable integration into the spliceosomal A complex—a poorly understood, multistep process that is facilitated by the DEAD-box helicase Prp5 (refs. 1,2,3,4). During this process, the U2 small nuclear RNA (snRNA) forms an RNA duplex with the pre-mRNA branch site (the U2–BS helix), which is proofread by Prp5 at this stage through an unclear mechanism5. Here, by deleting the branch-site adenosine (BS-A) or mutating the branch-site sequence of an actin pre-mRNA, we stall the assembly of spliceosomes in extracts from the yeast Saccharomyces cerevisiae directly before the A complex is formed. We then determine the three-dimensional structure of this newly identified assembly intermediate by cryo-electron microscopy. Our structure indicates that the U2–BS helix has formed in this pre-A complex, but is not yet clamped by the HEAT domain of the Hsh155 protein (Hsh155HEAT), which exhibits an open conformation. The structure further reveals a large-scale remodelling/repositioning of the U1 and U2 snRNPs during the formation of the A complex that is required to allow subsequent binding of the U4/U6.U5 tri-snRNP, but that this repositioning is blocked in the pre-A complex by the presence of Prp5. Our data suggest that binding of Hsh155HEAT to the bulged BS-A of the U2–BS helix triggers closure of Hsh155HEAT, which in turn destabilizes Prp5 binding. Thus, Prp5 proofreads the branch site indirectly, hindering spliceosome assembly if branch-site mutations prevent the remodelling of Hsh155HEAT. Our data provide structural insights into how a spliceosomal helicase enhances the fidelity of pre-mRNA splicing.

DOI: 10.1038/s41586-021-03789-5

Source: https://www.nature.com/articles/s41586-021-03789-5

Nature：《自然》，创刊于1869年。隶属于施普林格·自然出版集团，最新IF：69.504
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